The objective of this proposal is to prepare xenoantisera, in rabbits, reacting with defined membrane antigens of functional subclasses of human thymus-dependent lymphocytes (T cells). T lymphoblastoid lines, peripheral blood T cells, TM cells, and TG cells with initially be used to prepare immunogens. Blast and non-blast T cells, alloantigen-binding and non-binding T cells, all isolated from primary and secondary mixed lymphocyte responses, and T blast cells responding to PPD will subsequently be used to prepare immunogens. Animals will be immunized with whole viable cells, cell membranes, and detergent-solubilized cell membranes. Solubilized membranes will be purified prior to immunization by negative selection, initially by removing irrelevant antigens shared by T and B lymphocytes, using affinity columns with bound rabbit antibodies to B lymphoblastoid cell membranes. Antigens common to all T cells will eventually be removed from solubilized membranes of putative T cell subclasses prior to immunization, by affinity chromatography using antibodies to T lymphoblastoid cells. All antisera will be absorbed with B lymphoblastoid cells until they fail to react with them in complement-mediated cytotoxicity, and by indirect immunofluoresence. Antisera will be analysed serologically, by complement-mediated cytotoxicity and indirect immunofluoresence, on all the T cell populations used to prepare immunogens. They will be analysed functionally by their ability in complement-mediated lysis to eliminate TM and TG cells, cytotoxic T cells, and T cells stimulated in the MLR or by PPD to undergo DNA synthesis. The antisera will also be characterized in terms of the nature of the antigens with which they react, by immunoprecipitation of detergent-solubilized membranes from the T cell populations labeled internally or externally, followed by analysis using polyacrylamide gel electrophoresis in SDS and isoelectric focusing in 6 M urea. Electrophoretic variants of the antigens, and alloantigenic variants detectable serologically will ultimately be sought.